1. Field of the Invention
This invention is directed to a synthetic vaccine, to a composition useful in elliciting formation of antibodies in a host animal and to a composition useful as a diagnostic aid. More especially this invention is directed to a synthetic antigenic composition comprising a synthetic peptide and carrier. Still more especially this invention is directed specifically to the nature of the carrier for the synthetic peptide.
2. Discussion of Related Applications
In my co-pending applications referred to above I disclosed a new system for determining that portion of the protein of a natural antigen or allergen which is responsible for the antigenicity or allergenicity of the protein. More especially I defined a process for determining the specific sequences of amino acid of proteinaceous allergens or antigens which are causative of an immune response when compositions containing the same are injected into host animals.
Thus I disclose not only that method for determining the specific sequence of amino acids but a method of preparing synthetic antigens or allergens knowing the precise number and sequence of amino acids which must be present. I also disclose numerous synthetic vaccines comprising a short polypeptide supported on a carrier, the carrier considered to be of critical importance in providing the active portion of the synthetic peptide chain with sufficient size so that the entire synthetic antigen or synthetic allergen can be recognized by the immune system and evoke formation of the corresponding antibodies.
Specifically, my synthetic vaccine comprises a physiologically acceptable carrier in or on which is disposed a synthetic peptide residue containing a sequence of at least six amino acids corresponding to the sequence of such amino acids in a protein antigen or allergen with the greatest local average hydrophilicity of the antigen or allergen, said local hydrophilicity of said protein antigen or allergen being defined by and determined by:
A. assigning relative hydrophilicity values to the amino acids of the protein antigen or allergen in accordance with relative relationship of such amino acids as shown in the table below:
TABLE 1 ______________________________________ Amino Acid Hydrophilicity Value ______________________________________ Arginine 3.0 Aspartic Acid 3.0 .+-. 1 Glutamic Acid 3.0 .+-. 1 Lysine 3.0 Serine 0.3 Asparagine 0.2 Glutamine 0.2 Glycine 0.0 Proline -.5 .+-. 1 Threonine -0.4 Alanine -0.5 Histidine -0.5 Cysteine -1.0 Methionine -1.3 Valine -1.5 Isoleucine -1.8 Leucine -1.8 Tyrosine -2.3 Phenylalanine -2.5 Tryptophan -3.4 ______________________________________
B. determining the repetitive local average of hydrophilicity values at a plurality of points along the amino acid sequence:
C. determining from such local points of repetitive averages the points of greatest local average hydrophilicity; said composition being characterized by evoking a protective immunological response or by stimulation of antibody formation or decreased sensitivity to allergen when introduced into a host animal in the absence of the entire amino acid sequence of the protein antigen or allergen.
At the heart of the development there is the determination of a sequence of six amino acids which are critical to the production of the immunological response. In accordance with such earlier invention this is done with the foreknowledge of the amino acid sequence of an antigen or allergen, but if the same is unknown, then the amino acid sequence of the entire protein must first be determined. This can be done by known but laborious means.
Given the amino acid sequence of the entire protein antigen or allergen, the next objective is to determine the point along said molecule where there is greatest local average hydrophilicity. This is initially done by assigning relative hydrophilicity values in accordance with the table above to each amino acid in the protein. Thereafter, those values are repetitively averaged along the length of the protein. While such method is partially successful (working for some proteins, but not others) when averaging groups range in size from four to ten successively connected amino acids, it is preferred that in determining such local averages the hydrophilicity values of five to seven linearly connected amino acids be employed, especially six such amino acids. At a plurality of points along the amino acid chain of the protein, the local averages are determined (moving average, increment of one).
Once the repetitive local averages of the specific hydrophilicity values are determined, the precise point of greatest hydrophilicity can be easily located by inspection or determined graphically or otherwise. It has been discovered that the six amino acids providing the greatest local average hydrophilicity are the sequence of six amino acids which are critical to the production of the immunological response. Stated differently, it has been found that this sequence of six amino acids is present in an epitope of the protein, i.e. the sequence of amino acids recognized by and bound by an antibody with immunological specificity. Such epitope, is hereinafter designated as the "H-epitope" as it is the epitope of greatest local average hydrophilicity.
With this realization of the precise sequence of amino acids which accounts for H-epitope of a given protein antigen or allergen, one can form a synthetic vaccine in any number of ways.
The synthetic vaccine is prepared either by chemically synthesizing a chain of amino acids corresponding to the sequence of amino acids of the H-epitope or the H-epitope is obtained from a protein containing the same by selective lysis such as by splitting the protein by the use of enzymes. The amino acid chain containing the H-epitope so obtained either synthetically or from naturally occurring protein is thereafter disposed on a physiologically acceptable carrier, and the resultant composition is thereafter diluted with physiologically acceptable medium. The composition is then ready for introduction into a host animal.
It will be realized that the process of the invention is useful in the formation of synthetic vaccines of known and unknown, identified or unidentified, protein antigens or allergens, since the focus is upon the portion of the protein molecule which provides the H-epitope. Thus, the synthetic vaccine of the invention can contain H-epitopes of single or multiple known or unknown protein antigens or allergens. The synthetic vaccine can contain a plurality of H-epitopes of a single antigen or can contain a single H-epitope of a first antigen and an H-epitope of a second antigen or allergen. The synthetic vaccine can contain one or more H-epitopes of an antigen or allergen alone or in combination with one or more H-epitopes of a second antigen or allergen. In fact, the synthetic vaccine can contain as may epitopes corresponding to said sequence of six amino acids of greatest local average hydrophilicity as desired, and said epitopes can correspond to the sequence of six amino acids from a wide variety of antigens or allergens. The vaccine contains at least one H-epitope. This H-epitope can be co-present with other epitopes of the same or different antigens which are not H-epitopes, i.e., do not correspond to the point of greatest local average hydrophilicity of the antigen or allergen.
The process of the invention is useful in the formation of synthetic vaccines from antigens whose amino acid sequence has not heretofore been reported. The art well knows how to determine the amino acid sequence of a protein antigen or allergen. It remains, therefore, a simple matter in accordance with the invention to determine the H-epitope.
The synthetic vaccine can have H-epitopes of any protein antigen or allergen. The vaccine of the following protein antigens or allergens are particularly contemplated. Hepatitis B surface antigen, histocompatibility antigens, influenza hemagglutinin, fowl plague virus hemagglutinin, rag weed allergens Ra.sub.3 and Ra.sub.5 and the antigens of the following viruses: vaccinia, Epstein-Barr virus, polio, rubella, cytomegalovirus, small pox, herpes, simplex types I and II, yellow fever, and many others.
It can also alternatively or additionally have and H-epitope of a protein of any of the following parasites: organisms carrying malaria (P. Falciporum, P. Ovace, etc.). Schistosomiasis, Onchocerca Volvulus and other filiarial parasites, Trypanosomes, Leisbmania, Chagas disease, amoebiasis, hookworm, and the like. In addition, vaccines of the following bacterial are especially contemplated: leprosy, tuberculosis, syphilis, gonorrhea and the like.
Vaccines of the following viruses can be made by the process of the invention: Infectious ectromelia virus, Cowpox virus, Herpes simplex virus Infectious bovine rhinotracheitis virus, Equine rhinopneumonitis (equine abortion) virus, Malignant catarrh virus of cattle, Feline rhinotracheitis virus, Canine herpesvirus, Epstein-Barr virus (ass, with infectious mononucleosis and Burkitt lymphoma), Marek's disease virus, Sheep pulmonary adenomatosis (Jaagziekte) virus, Cytomegaloviruses, Adenovirus group, Human papilloma virus, Feline panleucopaenia virus, Mink enteritis virus, African horse sickness virus (9 serotypes), Blue tongue virus (12 serotypes), Infectious pancreatic necrosis virus of trout, Fowl sarcoma virus (various strains), Avian leukosis virus, visceral, Avian leukosis virus, erythroblastic, Avian leukosis virus, myeloblastic, Osteopetrosis virus, Newcastle disease virus, Parainfluenza virus 1, Parainfluenza virus 4, Mumps virus, Turkey virus, CANADA/58, Canine distemper virus, Measles virus, Respiratory syncytial virus, Myxovirus, Type A viruses, such as Human influenza viruses, e.g. Ao/PR8/34, Al/CAM/46, and A2/Singapore/1/57; Fowl plague virus; Type B viruses e.g. B/Lee/40; Rabies virus; Eastern equinine encephalitis virus; Venezuelan equine encephalitis virus; Western equine encephalitis virus; Yellow fever virus, Dengue type 1 virus (=type 6), Dengue type 2 virus (=type 5); Dengue type 3 virus; Dengue type 4 virus; Japanese encephalitis virus, Kyasanur Forest virus; Louping ill virus; Murray Valley encephalitis virus; Omsk haemorrhagic fever virus (types 1 and 11); St. Louis encephalitis virus; Human rhinoviruses, Foot-and-mouth disease virus; Poliovirus type 1; Enterovirus Polio 2; Enterovirus Polio 3; Avian infectious bronchitis virus; Human respiratory virus; Transmissible gastro-enteritis virus of swine; Lymphocytic choriomeningitis virus; Lassa virus; Machupo virus; Pichinde virus; Tacaribe virus; Papillomavirus.
Similarly, the synthetic vaccine can have an H-epitope of any protein allergen such as the rag weed allergens.
It is to be understood that the foregoing lists are not all-inclusive, but simply exemplary, since the heart of the invention resides in reliably and confidently predicting and determining the H-epitope.
In forming a synthetic vaccine according to the earlier invention, it is preferred to insure that the epitope has the steric configuration to be recognized by an antibody; that the given sequence of 6 amino acids have bonded thereto as part of the amino acid chain at least three amino acids on either side thereof, these three adjacent amino acids serving as auxiliary acids to insure the stabilization of the epitope so that it is readily recognized by and neutralized by an antibody.
In one of its simplest forms, that invention comprises a physiologically acceptable carrier on which is disposed a synthetic peptide residue of the designated epitope. This synthetic peptide residue has a chain length of minimally six amino acids, preferably twelve amino acids (considering the three amino acids on either side thereof) and can contain an infinitely long chain of amino acids or their components, which can be characterized by the presence of other epitopes of the same or different antigen or allergen. Where it is free of such additional chain with or without such additional epitopes, it generally does not have an amino acid chain exceeding 50 amino acids. Where a short chain is desired containing the desired epitope, it preferably does not have an amino acid chain length greater than 40, more especially not greater than 30 and more particularly not greater than 20 amino acids. Optimally the peptide residue has an amino acid chain length of 12 to 18 amino acids, preferably 12 to 15 amino acids, especially 12 amino acids.
In my earlier application I disclose numerous physiologically acceptable carriers for the peptide residue including those which are animal, vegetable and mineral. Specifically disclosed carriers included segments of polyamino acid, polysaccharides, polyamides, vinyl polymers, ester polymers, as well as proteins especially subclass hemoglobin, human serum proteins, tetanus toxoid.
One problem that exists in the field of vaccines relates to the nature of the carrier. Since optimally the vaccine stimulates production of only those antibodies specific to the antigen or allergen, the carrier should not evoke antibody formation to itself. The production of antibodies or any other substance in response to the carrier portion of the vaccine complicates the immune system's behavior, can be the cause of side reactions, and can compete with the production of antibodies to the synthetic antigen or allergen. It has therefore been desirable to provide a carrier for a synthetic vaccine where the carrier portion of the molecule is substantially inert to the immune system and does not evoke the production of antibodies specific thereto. It is the further object of this invention to provide an improved synthetic vaccine comprising a synthetic peptide residue disposed in or on a carrier where the carrier is one which is compatible with the organism into which the vaccine is to be introduced and can be readily metabolized by such host animal and in time be excreted without complications to the injection site or the various organs of the body.